Brassinosteroid biosynthesis inhibitors

ABSTRACT

Compound represented by the following formula (I):wherein R1 represents a lower alkyl group, R2 represents a phenyl group which may be substituted or a lower alkyl group and R3 represents a phenyl group which may be substituted (e.g., 4-(4-chlorophenyl)-2-phenyl-3-(1,2,4-triazoyl)-butan-2-ol) or salts thereof. The compounds have a specific inhibitory action against the brassinosteroid biosynthesis, and are useful as plant growth regulators.

This application is a 371 of PCT/J99/04380, filed on Sep. 12, 1999.

TECHNICAL FIELD

The present invention relates to a compound having inhibitory actionagainst the brassinosteroid biosynthesis and a plant growth regulatorcomprising said compound.

BACKGROUND ART

Brassinosteroids have recently recognized as a new class of planthormones through the combination of molecular genetics and researches onbiosyntheses (Yokota, Trends in Plant Sci., 2, pp.137-143, 1997). Sincethe chemistry of brassinosteroids was established, biological activitiesof these homologues have been extensively studied, and their notableactions on plant growth have been revealed, which include elongation ofstalks, growth of pollen tubes, inclination of leaves, opening ofleaves, suppression of roots, activation of proton pump (Mandava andAnnu. Rev. Plant Physiol. Plant Mol. Biol., 39, pp.23-52, 1988),acceleration of ethylene production (Schlagnhaufer et al., Physiol.Plant, 61, pp.555-558, 1984), differentiation of vessel elements(Iwasaki et al., Plant Cell Physiol., 32, pp.1007-1014, 1991; Yamamotoet al., Plant Cell Physiol., 38, pp.980-983, 1997), and cell extension(Azpiroz et al., Plant Cell, 10, pp.219-230, 1998).

Furthermore, mechanisms and regulations of physiological actions ofbrassinosteroids have been being revealed by variety of studies on theirbiosynthesis (Clouse, Plant J. 10, pp.1-8, 1996; Fujioka et al.,Physiol. Plant, 100, pp.710-715, 1997). At present, 40 or morebrassinosteroids have been identified. Most of C28-brassinosteroids arecommon vegetable sterols, and they are considered to be biosynthesizedfrom campesterol which has the same carbon side chain as that ofbrassinolide.

Some Arabidopsis mutants which show characteristic dwarfism have beenisolated, i.e., dwfl: Feldman et al., Science, 243, pp.1351-1354, 1989;dim: Takahashi et al., Genes Dev., 9, pp.97-107, 1995; cbb1: Kauschmannet al., Plant J., 9, pp.701-703, 1996. Their structuralphotomorphogenesis and dwarfism (cpd; Szekeres et al., Cell, 85,pp.171-182, 1997) and de-etiolation (det2: Li et al., Science, 272,pp.398-401, 1996; Fujioka et al., Plant Cell, 9, pp.1951-1962, 1997) areknown. The mutants have deficiencies in the brassinosteroid biosyntheticpathway. Further, a dwarf mutant of Pisum sativum was recentlycharacterized, and the mutant was reported as a brassinosteroiddeficient mutant (Nomura et al., Plant Physiol., 113, pp.31-37, 1997).In these plants, use of brassinolide is known to negate severe dwarfismof the mutants. Although these findings suggest that roles ofbrassinosteroids are indispensable for growth and development of plants,an effective tool other than the analysis of mutants has been desired toelucidate physiological importance of brassinolide.

As seen in researches of gibberellin action, specific inhibitors againstthe biosynthesis are generally very effective tools for elucidatingphysiological functions of endogenous substances. Specific inhibitorsfor the brassinosteroid biosynthesis are expected to provide a new toolfor understanding the functions of brassinosteroids. Uniconazol is apotent plant growth regulator (PGR) which inhibits the oxidationemployed by cytochrome P-450 in the steps of the gibberellinbiosynthesis from ent-kaurene to ent-kaurenoic acid. Yokota et al.observed slight reduction of the amount of endogenous castasterone as aside effect of that compound (Yokota et al., “Gibberellin”, SpringerVerlag, N.Y., pp.339-349, 1991). Although uniconazole inhibitsdifferentiation of vessel elements induced by brassinolide (Iwasaki etal., Plant Cell Physiol., 32, pp.1007-1014, 1991), its inhibitory actionagainst brassinolide is considered to be no more than an incidentalaction, because uniconazol essentially inhibits the gibberellinbiosynthesis.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a specific inhibitoragainst the brassinosteroid biosynthesis. Some mutants which aredeficient in enzymes for biosynthesis are known for Arabidopsis, andtheir morphologic changes are unique to mutants with deficiency in thebrassinosteroid biosynthesis. Therefore, the inventors of the presentinvention conducted intensive search for a compound inducing themorphologic changes unique to the mutants with the brassinosteroidbiosynthesis deficiency to find a specific inhibitor against thebrassinosteroid biosynthesis. As a result, they found that the triazolecompounds represented by the following formula (I) had the desiredinhibitory action. The present invention was achieved on the basis ofthese findings.

The present invention thus provides a compound represented by thefollowing formula (I):

wherein R¹ represents a lower alkyl group, R² represents a phenyl groupwhich may be substituted or a lower alkyl group, and R³ represents aphenyl group which may be substituted, or a salt thereof. According to apreferred embodiment of the present invention, there are provided theaforementioned compound or a salt thereof wherein R¹ is methyl group orethyl group, and R² represents a phenyl group which may be substitutedor tert-butyl group.

As another aspect of the present invention, there are provided aninhibitor against the brassinosteroid biosynthesis which comprises thecompound represented by the aforementioned formula (I) or aphysiologically acceptable salt thereof. The inhibitor of the presentinvention can be used as a plant growth regulator for, for example,suppression of plant elongation, suppression of pollen growth, retentionof freshness of flowers, anti-stress agents for plants, weeds control,suppression of plant retrogradation, hypertrophism of roots and soforth.

According to further aspects of the present invention, there areprovided a method for regulating plant growth by using the compoundrepresented by the aforementioned formula (I) or a salt thereof; and useof the compound represented by the aforementioned formula (I) or a saltthereof for the manufacture of the aforementioned plant growthregulator.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows actions of RMB-61 (1 μg/plant) on various brassinosteroids(BR, 0.1 ng/plant) observed in the explantation method. In the figure,“Angle” indicates an inclination angle; and results are indicated with“Cont” for control (no treatment); “BL” for brassinolide (0.1 ng); “CS”for castasterone (1 ng); “TY” for typhasterol (10 ng); “3DT” for3-dehydroteasterone (10 ng); “TE” for teasterone (100 ng); and “CT” forcathasterone (100 ng) wherein each dose per plant is shown. The darkshaded columns (left) show the results obtained with BR alone, and thelight shaded columns (right) show the results obtained with BR+RMB-61.

FIG. 2 shows actions of RMB-61 (5 μg/plant) observed in the whole plantmethod. In the figure, “Angle” indicates an inclination angle; and theresults are indicated with “Cont” for control (no treatment); “CT” forcathasterone (100 ng); “TE” for teasterone (100 ng); and “BL” forbrassinolide (0.1 ng) wherein each dose per plant is shown.

BEST MODE FOR CARRYING OUT THE INVENTION

In the aforementioned formula (I), R¹ represents a lower alkyl group. Asthe lower alkyl group, a linear or branched alkyl group having 1 toabout 6 carbon atoms can be used. Examples include methyl group, ethylgroup, n-propyl group, isopropyl group, n-butyl group, sec-butyl group,tert-butyl group and so forth. The lower alkyl group represented by R¹is preferably methyl group or ethyl group.

R² represents a phenyl group which may be substituted or a lower alkylgroup. As the lower alkyl group represented by R², a linear or branchedalkyl group having about 3 to about 6 carbon atoms is preferred, and abulky alkyl group such as isopropyl group and tert-butyl group ispreferred. When the phenyl group represented by R² or R³ is substituted,types, numbers and substituting positions of substituents are notparticularly limited. For example, the phenyl group may have preferably1 to 3, more preferably 1 or 2 of substituents. Where the phenyl grouphas 2 or more substituents, they may be the same or different.

Examples of the substituent on the phenyl group include, for example, ahalogen atom (any of fluorine atom, chlorine atom, bromine atom andiodine atom), a lower alkyl group (methyl group, ethyl group and thelike), a lower cycloalkyl group (cyclopropyl group and the like), ahalogenated lower alkyl group (trifluoromethyl group and the like), alower alkoxy group (methoxy group, ethoxy group and the like), aminogroup, mono- or dialkylamino group, carboxyl group, an alkoxycarbonylgroup (ethoxycarbonyl group and the like), an alkanoyl group (acetylgroup and the like), an aroyl group (benzoyl group and the like), anaralkyl group (benzyl group and the like), an aryl group (phenyl groupand the like), a heteroaryl group (pyridyl group and the like),heterocyclic group (pyrrolidinyl group and the like), hydroxyl group,nitro group, cyano group and so forth. However, the substituents are notlimited to these examples. Among them, a halogen atom, a lower alkylgroup, a halogenated lower alkyl group, a lower alkoxy group and soforth are preferred.

The compounds of the present invention have two asymmetric carbon atomsin the fundamental structure, and may have one or more furtherasymmetric carbon atoms depending on the type of the substituent.Optically active compounds and diastereoisomers in pure forms based onthe asymmetric carbon atoms as well any mixtures of the isomers (forexample, mixtures of two or more of diastereoisomers), racemates and soforth fall within the scope of the present invention. Further, thecompounds of the present invention may form acid addition salts, and mayfurther form acid addition salts depending on the type of thesubstituent. The types of the salts are not particularly limited, andexamples of the salts include salts with mineral acids such ashydrochloric acid, and sulfuric acid, salts with organic acids such asp-toluenesulfonic acid, methanesulfonic acid, and tartaric acid, metalsalts such as sodium salts, potassium salts, and calcium salts, ammoniumsalts, salts with organic amines such as triethylamine, salts with aminoacids such as glycine and so forth.

Examples of the compounds of the present invention will be mentionedbelow. However, the compounds of the present invention are not limitedto these examples. The symbols in the table represent: Me: methyl group;Et: ethyl group; MeO: methoxy group; tert-Bu: tertiary butyl group; andPh: phenyl group. As for substituted phenyl groups, 4-Cl-Ph represents4-chlorophenyl group; 2,4-di-Cl-Ph represents 2,4-dichlorophenyl group;2-Cl-4-F-Ph represents 2-chloro-4-fluorophenyl group and so forth, andothers are represented similarly.

TABLE 1 Compound Number R¹ R² R^(a)  1 (Diastereomer mixture) Me Ph4-Cl-Ph  2 (Isomer I) Me Ph 4-Cl-Ph  3 (Isomer II) Me Ph 4-Cl-Ph  4 MePh 4-Br-Ph  5 Me Ph 4-Br-Ph  6 Me Ph 2,4-di-Cl-Ph  7 Me Ph 3,4-di-Cl-Ph 8 Me Ph 4-Me-Ph  9 Me Ph 4-CF₃-Ph 10 Me Ph 3-Cl-Ph 11 Me Ph 3-MeO-Ph 12(Isomer I) Me 4-Cl-Ph 4-Cl-Ph 13 (Isomer II) Me 4-Cl-Ph 4-Cl-Ph 14 Me4-Cl-Ph 2,4-di-Cl-Ph 15 (Isomer I) Me 4-F-Ph 4-Cl-Ph 16 (Isomer II) Me4-F-Ph 4-Cl-Ph 17 Me 4-F-Ph 2,4-di-Cl-Ph 18 Me 4-F-Ph 4-Me-Ph 19 (IsomerI) Me 4-MeO-Ph 4-Cl-Ph 20 (Isomer II) Me 4-MeO-Ph 4-Cl-Ph 21 Me 4-MeO-Ph2,4-di-Cl-Ph 22 Et Ph 4-Cl-Ph 23 (Isomer I) Me tert-Bu 4-Cl-Ph 24(Isomer II) Me tert-Bu 4-Cl-Ph 25 Me tert-Bu 2,4-di-Cl-Ph 26 Me Ph4-F-Ph 27 Me Ph 2,4-di-F-Ph 28 Me Ph 2-Cl-4-F-Ph 29 Me 4-Cl-Ph 4-Br-Ph30 Me 4-Cl-Ph 3,4-di-Cl-Ph 31 Me 4-Cl-Ph 4-Me-Ph 32 Me 4-Cl-Ph 4-CF₃-Ph33 Me 4-Cl-Ph 3-Cl-Ph 34 Me 4-Cl-Ph 3-MeO-Ph 35 Me 4-Cl-Ph 4-F-Ph 36 Me4-Cl-Ph 2,4-di-F-Ph 37 Me 4-Cl-Ph 2-Cl-4-F-Ph 38 Me 4-F-Ph 4-Br-Ph 39 Me4-F-Ph 3,4-di-Cl-Ph 40 Me 4-F-Ph 4-CF₃-Ph 41 Me 4-F-Ph 3-Cl-Ph 42 Me4-F-Ph 4-F-Ph 43 Me 4-F-Ph 3-MeO-Ph 44 Me 4-F-Ph 2,4-di-F-Ph 45 Me4-F-Ph 2-Cl-4-F-Ph 46 Et Ph 2,4-di-Cl 47 Et Ph 4-Br-Ph 48 Et Ph 4-Me-Ph49 Et Ph 3,4-di-Cl-Ph 50 Et Ph 4-CF₃-Ph 51 Et Ph 3-Cl-Ph 52 Et Ph3-MeO-Ph 53 Et Ph 4-F-Ph 54 Et Ph 2,4-di-F-Ph 55 Et Ph 2-Cl-4-F-Ph 56 Et4-Cl-Ph 2,4-di-Cl-Ph 57 Et 4-Cl-Ph 4-Br-Ph 58 Et 4-Cl-Ph 3,4-di-Cl-Ph 59Et 4-Cl-Ph 4-Me-Ph 60 Et 4-Cl-Ph 4-CF₃-Ph 61 Et 4-Cl-Ph 3-Cl-Ph 62 Et4-Cl-Ph 3-MeO-Ph 63 Et 4-Cl-Ph 4-F-Ph 64 Et 4-Cl-Ph 2,4-di-F-Ph 65 Et4-Cl-Ph 2-Cl-4-F-Ph 66 Et 4-F-Ph 4-Cl-Ph 67 Et 4-F-Ph 2,4-di-Cl-Ph 68 Et4-F-Ph 4-Br-Ph 69 Et 4-F-Ph 3,4-di-Cl-Ph 70 Et 4-F-Ph 4-Me-Ph 71 Et4-F-Ph 4-CF₃-Ph 72 Et 4-F-Ph 3-Cl-Ph 73 Et 4-F-Ph 3-MeO-Ph 74 Et 4-F-Ph4-F-Ph 75 Et 4-F-Ph 2,4-di-F-Ph 76 Me 2,4-di-Cl-Ph 4-Cl-Ph 77 Me2,4-di-Cl-Ph 2,4-di-Cl-Ph 78 Me 2,4-di-Cl-Ph 4-Br 79 Me 2,4-di-Cl-Ph3,4-di-Cl-Ph 80 Me 2,4-di-Cl-Ph 4-Me-Ph 81 Me 2,4-di-Cl-Ph 4-CF₃-Ph 82Me 2,4-di-Cl-Ph 3-Cl 83 Me 2,4-di-Cl-Ph 3-MeO-Ph 84 Me 2,4-di-Cl-Ph4-F-Ph 85 Me 2,4-di-Cl-Ph 2,4-di-F-Ph 86 Et 2,4-di-Cl-Ph 4-Cl-Ph 87 Et2,4-di-Cl-Ph 2,4-di-Cl-Ph 88 Et 2,4-di-Cl-Ph 4-Br 89 Et 2,4-di-Cl-Ph3,4-di-Cl-Ph 90 Et 2,4-di-Cl-Ph 4-Me-Ph 91 Et 2,4-di-Cl-Ph 4-CF₃-Ph 92Et 2,4-di-Cl-Ph 3-Cl 93 Et 2,4-di-Cl-Ph 3-MeO-Ph 94 Et 2,4-di-Cl-Ph4-F-Ph 95 Et 2,4-di-Cl-Ph 2,4-di-F-Ph 96 Et 2,4-di-Cl-Ph 2-Cl-4-F-Ph

Methods for preparing the compounds of the present invention are notparticularly limited. For example, the compounds can be produced by thefollowing method. In the examples of the specification, the preparationsof typical compounds of the present invention will be specificallyexplained in detail. Therefore, those skilled in the art can readilyprepare the compounds of the general formula (I) by referring to thefollowing general descriptions and specific explanations in the examplesand appropriately choosing compounds as starting materials, reagents,reaction conditions and so forth, and by optionally adding suitablemodifications and alterations to these methods. The reactive functionalgroups of compounds as starting materials or reagents may be protectedwith suitable protective groups, if necessary. Such protective groupscan be appropriately chosen by those skilled in the art depending on thetypes of functional groups.

Compound (III) can be produced by reacting Compound (II) with1,2,4-triazole or an alkali metal salt thereof in a suitable solventsuch as acetone, acetonitrile, methanol, ethanol, and dimethylformamidein the presence of a suitable acid trapping agent such as potassiumcarbonate, sodium carbonate, potassium butoxide, sodium hydride,potassium hydride, sodium methoxide, and sodium ethoxide (in the scheme,Xn represents one or more substituents such as hydrogen atom, chlorineatom, fluorine atom, methoxy group, and Hal represents chlorine atom,bromine atom, or iodine atom).

Compound (IV) can be prepared by reacting Compound (III) with a benzylhalide derivative in a suitable dry solvent such as methanol, ethanol,tetrahydrofuran, and dimethylformamide in the presence of a suitablebase such as potassium butoxide, sodium hydride, potassium hydride,sodium methoxide, and sodium ethoxide (in the scheme, Yn represents oneor more substituents such as chlorine atom, bromine atom, methyl group,trifluoromethyl group, and methoxy group).

Compound (I) of the present invention can be prepared by reacting anorganometallic compound such as alkyl lithium and Grignard reagents withCompound (IV) in an aprotic anhydrous solvent such as tetrahydrofuran,dimethylformamide, diglim, dioxane, and diethyl ether (in the scheme, R¹has the same meaning as that defined in the formula (I), and preferablyrepresents methyl group or ethyl group, and Xn and Yn have the samemeanings as those defined above). In the alkylation of the abovereaction, an alkyl lithium or an alkyl magnesium bromide may sometimesgive a different ratio of stereoisomers as reaction products. Wheremethylation is carried out, respective reagents may sometimes give adifferent diastereoisomer in an approximately 100% yield. Therefore, itis possible to selectively prepare a desired stereoisomer by suitablychoosing a reagent.

The compounds of the present invention or salts thereof have specificinhibitory action against the brassinosteroid biosynthesis. Therefore,the compounds of the present invention or salts thereof are useful as,for example, active ingredients of plant growth regulators. The term“plant growth regulation” used in this specification should be construedin its broadest sense, including, for example, dwarfing of plants(suppression of plant elongation), pollen growth inhibition, retentionof flower freshness, use of plant anti-stress agents (heat, dryness,coldness or the like), weed control by regulation of reproduction,suppression of plant retrogradation, control of hypertrophy of root andso forth. For example, plant growth dwarfing agents, plant growthretardants, herbicides and so forth are typical examples of the plantgrowth regulators of the present invention. However, the plant growthregulators of the present invention are not limited to these examples.

The plant growth regulators of the present invention can be formulated,for example, as an agricultural composition by using formulationadditives well known in the art. Forms of the agricultural compositionare not particularly limited, and any forms that can be used in the artmay be chosen. For example, compositions in the forms of emulsions,liquids, oils, water soluble powders, wettable powders, flowables,powders, subtilized granules, granules, aerosols, fumigants, pastes andso forth can be used. The methods for manufacturing the agriculturalcomposition are also not particularly limited, and any methods availableto those skilled in the art can be appropriately employed. As the activeingredient of the plant growth regulators of the present invention, twoor more of the compounds represented by the aforementioned formula (I)or salts thereof may be used in combination. Further, other activeingredients of agricultural chemicals such as insecticides, fungicides,insecticidal and fungicidal agents, herbicides and the like. Methods ofapplication and doses of the plant growth regulators of the presentinvention can be suitably chosen by those skilled in the art dependingon conditions including a purpose of application, a dosage form, a plotto be treated and so forth.

EXAMPLES

The present invention will be explained more specifically with referenceto examples. However, the scope of the present invention is not limitedto the following examples.

Example 1 Preparation of the Compound of the Present Invention

To 20 ml of dimethylformamide, 1.99 g of bromoacetophenone, 0.69 g oftriazole and 2 g of potassium carbonate were added, and the mixture wasallowed to react at room temperature for 16 hours. The reaction mixturewas poured into 100 ml of water, and the deposited crystals wereseparated by filtration. The crystals obtained were recrystallized fromhexane/ethylacetate to obtain 2-(1,2,4-triazoyl)acetophenone (yield:85%).

In 50 ml of dry dimetbylformamide, 1.87 g of2-(1,2,4-triazoyl)acetophenone was dissolved, and the solution was addedwith 0.48 g of 60% sodium hydride and stirred for 10 minutes with icecooling. The reaction mixture was added dropwise with 1.61 g of4-chlorobenzyl chloride dissolved in dimethylformamide, and then stirredfor 2 hours. The reaction was stopped by adding 1 ml of methanol to thereaction mixture, and the solvent was evaporated under reduced pressure.The residue was distributed between water and ether, and the ether layerwas separated. The aqueous layer was extracted twice with ether, and theether layers were combined with the ether layer previously obtained andwashed with saturated brine. The ether layer was dried over anhydrousmagnesium sulfate, and then the solvent was evaporated under reducedpressure. The residue was purified by silica gel column chromatography(hexane/ethyl acetate) to obtain2-(4-chlorobenzyl)-2-(1,2,4-triazoyl)acetophenone (yield: 68%).

Under a nitrogen flow, 0.312 g of2-(4-chlorobenzyl)-2-(1,2,4-triazoyl)-acetophenone was dissolved in 10ml of dry tetrahydrofuran. This solution was cooled to −80° C. with dryice/acetone, and added dropwise with 1.1 equivalents of an ethersolution of methyl lithium with stirring. The reaction mixture wasstirred for 30 minutes, and returned to room temperature. The reactionmixture was poured into an ammonium chloride solution, and extractedthree times with ethyl acetate. The organic phase was washed withsaturated brine, and dried over anhydrous magnesium sulfate, and thesolvent was evaporated under reduced pressure. The residue was purifiedby silica gel column chromatography (hexane/ethyl acetate) to obtain4-(4-chlorophenyl)-2-phenyl-3-(1,2,4-triazoyl)butan-2-ol (Compound 1, amixture of diastereomers, yield: 58%).

The above product was a mixture of two diastereomers (80:20), and eachdiastereomer was a mixture of equal amounts of enantiomers. Each of thediastereomers can be separated by the aforementioned columnchromatography.

¹H-NMR (δ, ppm, CDCl₃).

Diastereomer (I) (Compound 2)

1.28 (s, 3H), 2.70 (dd, 1H, J=2.6, 14.3 Hz), 3.24 (dd, 1H, J=11.5, 14.3Hz), 4.38 (s, 1H), OH), 4.42 (dd, 1H, J=2.3, 11.5 Hz), 6.54 (d, 2H,J=8.4 Hz), 7.07 (d, 2H, J=8.4 Hz), 7.32-7.60 (m, 5H), 7.67 (s, 1H), 8.06(s, 1H).

Diastereomer (II) (Compound 3)

1.62 (s, 3H), 3.33 (dd, 1H, J=3.19, 14.1 Hz), 3.499 (dd, 1H, J=11.5,14.1 Hz), 4.495 (dd, 1H, J=2.9, 11.5 Hz), 4.71 (s, 1H, OH), 6.78 (d, 2H,J=8.4 Hz), 7.13 (d, 2H, J=8.4 Hz), 7.10-7.26 (m, 5H), 7.236 (s, 1H),7.799 (s, 1H)

Isomer (I) can be selectively prepared by using methyl magnesium bromideinstead of methyl lithium in the aforementioned reaction.

Under a nitrogen flow, 0.312 g of2-(4-chlorobenzyl)-2-(1,2,4-triazoyl)-acetophenone was dissolved in 10ml of dry tetrahydrofuran. This solution was cooled to −80° C. with dryice/acetone, and added dropwise with 1.1 equivalents of an ethersolution of methyl magnesium bromide with stirring. The reaction mixturewas stirred for 30 minutes, and returned to room temperature. Thereaction mixture was poured into an ammonium chloride solution, andextracted three times with ethyl acetate. The organic phase was washedwith saturated brine, and dried over anhydrous magnesium sulfate, andthe solvent was evaporated under reduced pressure. The residue waspurified by silica gel column chromatography (hexane/ethyl acetate) toobtain 4-(4-chlorophenyl)-2-phenyl-3-(1,2,4-triazoyl)butan-2-ol (totalyield: 75%).

Each of the aforementioned Isomer (I) and Isomer (II) was a mixture oftwo enantiomers (1:1). These enantiomers were resolved by using anoptical resolution column (Daicel Chemical Industries, Ltd., ChiralcellOJ), and four stereoisomers (2a, 2b/3a, 3b) were finally obtained.Respective NMR spectra of the stereoisomers were the same as those ofIsomer (I) and Isomer (II).

NMR spectrum data of the compounds prepared in a similar manner areshown below (δ, ppm, CDCl₃, the compound numbers correspond to thosementioned in the above table).

Compound 2, Compound 2a and Compound 2b (Compounds 2a and 2b areenantiomers of Compound 2, the same shall apply hereinafter)

1.28 (s, 3H), 2.70 (dd, 1H, J=2.6, 14.3 Hz), 3.24 (dd, 1H, J=11.5, 14.3Hz), 4.38 (s, 1H, OH), 4.42 (dd, 1H, J=2.3, 11.5 Hz), 6.54 (d, 2H, J=8.4Hz), 7.07 (d, 2H, J=8.4 Hz), 7.32-7.60 (m, 5H), 7.67 (s, 1H), 8.06 (s,1H).

Compound 3, Compound 3a and Compound 3b

1.62 (s, 5H), 3.33 (dd, 1H, J=3.19, 14.1 Hz), 3.499 (dd, 1H, J=11.5,14.1 Hz), 4.495 (dd, 1H, J=2.9, 11.5 Hz), 4.71 (s, 1H, OH), 6.78 (d, 2H,J=8.4 Hz), 7.13 (d, 2H, J=8.4 Hz), 7.10-7.26 (m, 5H), 7.236 (s, 1H),7.799(s, 1H).

Compound 4

1.283 (s, 3H), 2.678 (dd, 1H, J=2.7, 14.3 Hz), 3.212 (dd, 1H, J=11.5,14.3 Hz), 4.368 (s, 1H, OH), 4.417 (dd, 1H, J=2.4, 11.7 Hz), 6.48 (d,2H, J=8.4 Hz), 7.221 (d, 2H, J=8.4 Hz), 7.48-7.34 (m, 5H), 7.675 (s,1H), 8.059 (s, 1H).

Compound 5, Compound 5a and Compound 5b

1.771 (s, 3H), 3.317 (dd, 1H, J=3,14.1 Hz), 3.492 (dd, 1H, J=11.5, 14.1Hz), 4.492 (dd, 1H), J=3, 11.5 Hz), 4.706 (s, 1H, OH), 6.725 (d, 2H,J=8.27 Hz), 7.298-7.138 (m, 8H), 7.805 (s, 1H).

Compound 6

1.294 (s, 3H), 2.917 (dd, 1H, J=2.9, 14.2 Hz), 3.265 (dd, 1H, J=11.6,14.2 Hz), 4.477 (s, 1H, OH), 4.670 (dd, 1H, J=2.9, 11.5 Hz), 6.416 (d,1H, J=8.3 Hz), 6.890 (dd, 1H, J=2.0, 8.3 Hz), 7.245 (d, 1H, J=2.0 Hz),7.633-7.319 (m, 5H), 7.713 (s, 1H), 8.043 (s, 1H).

Compound 7

1.286 (s, 3H), 2.687 (dd, 1H, J=2.8, 14.4 Hz), 3.244 (dd, 1H, J=11.5,14.4 Hz), 4.285 (s, 1H, OH), 4.433 (dd, 1H, J=2.7, 11.5 Hz), 6.412 (dd,1H, H=2.0, 8.2 Hz), 6.763 (d, 1H, J=2.0 Hz), 7.151 (d, 1H, J=8.2 Hz),7.60-7.34 (m, 5H), 7.734 (s, 1H), 8.073 (s, 1H).

Compound 8

1.285 (s, 3H), 2.216 (s, 3H), 2.675 (dd, 1H, J=2.7, 14.3 Hz), 3.171 (dd,1H, J=11.6, 14.2 Hz), 4.436 (dd, 1H, J=2.5, 11.6 Hz), 4.525 (s, 1H, OH),6.485 (d, 2H, J=7.8 Hz), 6.90 (d, 2H, J=7.8 Hz), 7.616-7.327 (m, 5H),7.629 (s, 1H), 8.054 (s, 1H).

Compound 9

1.293 (s, 3H), 2.239 (dd, 1H, J=2.2, 14.3 Hz), 3.338 (dd, 1H, J=11.5,14.2 Hz), 4.336 (s, 1H, OH), 4.465 (dd, 1H, J=2.5, 11.5 Hz), 6.734 (d,2H, J=8 Hz), 7.360 (d, 2H, J=8 Hz), 7.613-7.346 (m, 5H), 7.682 (s, 1H),8.074 (s, 1H).

Compound 10

1.288 (s, 3H), 2.703 (dd, 1H, J=2.4, 14.2 Hz), 3.237 (dd, 1H, J=11.6,14.2 Hz), 4.399 (s, 1H, OH), 4.456 (dd, 1H, J=2.6, 11.6 Hz), 6.457 (d,1H, J=7.5 Hz), 6.666 (s, 1H), 6.989-7.102 (m, 2H), 7.611-7.338 (m, 5H),7.696 (s, 1H), 8.070 (s, 1H).

Compound 11

1.288 (s, 3H), 2.696 (dd, 1H, J=2.4,14.6 Hz), 3.187 (dd, 1H, J=11.5,14.2 Hz), 3.638 (s, 3H), 4.465 (dd, 1H, J=2.4, 11.5 Hz), 4.509 (s, 1H),6.113 (s, 1H), 6.213 (d, 1H, J=7.68 Hz), 6.642 (dd, 1H, J=2.4, 8.2 Hz),7.024 (t, 1H, J=7.9 Hz), 7.615-7.326 (m, 5H), 7.664 (s, 1H), 8.066 (s,1H).

Compound 12

1.262 (s, 3H), 2.649 (dd, 1H, J=2.6, 14.4 Hz), 3.213 (dd, 1H, J=11.7,14.2 Hz), 4.358 (dd, 1H, J=2.5, 11.5 Hz), 4.499(s, 1H, OH), 6.534 (d,2H, J=8.3 Hz), 7.077 (d, 2H, J=8.3 Hz), 7.429 (d, 2H, J=8.4 Hz), 7.537(d, 2H, J=8.4 Hz), 7.648 (s, 1H), 8.065 (s, 1H).

Compound 13, Compound 13a and Compound 13b

1.738 (s, 3H), 3.321 (dd, 1H, J=3.32, 14.1 Hz), 3.469 (dd, 1H, 11.4,14.1 Hz), 4.459 (dd, 1H, J=3.1, 11.4 Hz), 4.848 (s, 1H, OH), 6.764 (d,2H, J=8.4 Hz), 7.130-7.230 (m, 7H), 7.810 (s, 1H).

Compound 14

1.265 (s, 3H), 2.886 (dd, 1H, J=3.0, 14.2 Hz), 3.248 (dd, 1H, J=11.6,14.2 Hz), 4.582 (s, 1H, OH), 4.6338(dd, 1H, 3.0, 11.4 Hz), 6.409 (d, 1H,J=8.3 Hz), 6.90 (dd, 1H, J=2.1, 8.3 Hz), 7.179 (d, 1H, J=2.7 Hz), 7.414(d, 2H, J=8.7 Hz), 7.568 (d, 2H, J=8.72 Hz), 7.687 (s, 1H), 8.05 (s,1H).

Compound 15

1.274 (s, 3H), 2.670 (d, 1H, J=2.7, 14.3 Hz), 3.218 (dd, 1H, J=11.7,14.3 Hz), 4.359 (dd, 1H, 2.4, 11.5 Hz), 4.457 (s, 1H, OH), 6.537 (d, 2H,J=8.4 Hz), 7.078 (d, 2H, J=8.4 Hz), 7.144 (t, 2H, J=8.5 Hz), 7.569 (dd,2H), 7.649 (s, 1H), 8.065 (s, 1H).

Compound 16

1.754 (s, 3H), 3.343 (dd, 1H, J=3.2, 11.5 Hz), 3.50 (dd, 1H, J=11.5,14.2 Hz), 4.478 (dd, 1H, J=3.0, 14.4 Hz), 4.488 (s, 1H, OH), 6.779-7.286(m, 9H), 7.814 (s, 1H).

Compound 17

1.276 (s, 3H), 2.903 (dd, 1H, J=3.0, 14.2 Hz), 3.245 (dd, 1H, J=11.5,14.2 Hz), 4.544 (s, 1H, OH), 4.614 (dd, 1H, J=3.0,11.5 Hz), 6.402 (d,1H, J=8.3 Hz), 6.871 (dd, 2H, J=2, 8.5 Hz), 7.125 (t, 2H, J=8.8 Hz),7.594 (dd, 2H, J=5.2, 8.9 Hz), 7.688 (s, 1H), 8.049 (s, 1H).

Compound 18

1.272 (s, 3H), 2.219 (s, 3H), 2.653 (dd, 1H, J=2.5, 14.2 Hz), 3.159 (dd,1H, J=11.5, 14.2 Hz), 4.374 (dd, 1H, J=2.3, 11.5 Hz), 4.596 (s, 1H, OH),6.476 (d, 2H, J=8.0 Hz), 6.905 (d, 2H, J=7.7 Hz), 7.155 (d, 2H, J=8.0Hz), 7.576 (dd, 2H, J=5.3, 8.9 Hz), 7.607 (s, 1H), 8.053 (s, 1H).

Compound 19

1.269 (s, 3H), 2.739 (dd, 1H, J=2.6, 14.3 Hz), 3.212 (dd, 1H, J=11.5,14.2 Hz), 3.854 (s, 3H), 4.335 (s, 1H), 4.361 (dd, 1H, J=2.6, 11.5 Hz),6.556 (d, 2H, J=8.4 Hz), 6.972 (d, 2H), J=8.9 Hz), 7.069 (d, 2H, J=8.4Hz), 7.489 (d, 2H, J=8.9 Hz), 7.654 (s, 1H), 8.042 (s, 1H).

Compound 20

1.745 (s, 3H), 3.301 (dd, 1H, J=3.1, 14.1 Hz), 3.486 (dd, 1H, J=11.6,14.1 Hz), 3.723 (s, 3H), 4.452 (dd, 1H, J=3.1, 11.6 Hz), 4.560 (s, 1H,OH), 6.724 (m, 4H), 7.187 (m, 5H), 7.821 (s, 1H).

Compound 21

1.274 (s, 3H), 2.957 (dd, 1H, J=2.9, 14.2 Hz), 3.251 (dd, 1H, J=11.5,14.1 Hz), 3.848 (s, 3H), 4.416 (s, 1H), 4.623 (dd, 1H, 2.9, 11.5 Hz),6.427 (d, 1H, J=8.2 Hz), 6.957 (d, 2H), J=8.4 Hz), 7.047-6.821 (m, 2H),7.522 (d, 2H, J=8.4 Hz), 7.694 (s, 1H), 8.030 (s, 1H).

Compound 22

0.593 (t, 3H), 1.09 (q, 1H, J=7.14 Hz), 1.68 (q, 1H, J=7.3 Hz), 2.64(dd, 1H, J=2.74, 14.5 Hz), 3.225 (dd, 1H, J=11.5, 14.4 Hz), 4.201 (s,1H), 4.44 (dd, 1H, J=2.4, 11.5 Hz), 6.523 (d, 2H, J=8.4 Hz), 7.066 (d,2H, J=8.4 Hz), 7.56-7.30 (m, 5H), 7.680 (s, 1H), 8.063 (s, 1H).

Compound 23

0.898 (s, 9H), 1.352 (s, 3H), 2.133 (s, 1H, OH), 3.249 (dd, 1H, J=11.5,13.6 Hz), 3.454 (dd, 1H, J=2.5, 13.6 Hz), 4.374 (dd, 1H, J=2.5, 11.5Hz), 6.713 (d, 2H, J=8.3 Hz), 7.115 (d, 2H), J=8.3 Hz), 7.612 (s, 1H),7.950 (s, 1H).

Compound 24

0.833 (s, 9H), 1.456 (s, 3H), 3.228 (dd, 1H, J=3.6, 13.8 Hz), 3.332 (dd,1H, J=11.3, 13.8 Hz), 3.437 (s, 1H), 4.466 (dd, 1H, J=3.6, 11.3 Hz),6.747 (d, 2H, J=8.3 Hz), 7.138 (d, 2H, J=8.3 Hz), 7.529 (s, 1H), 7.935(s, 1H).

Compound 25

0.780 (s, 9H), 1.490 (s, 3H), 3.315 (dd, 1H, J=11.7, 13.7 Hz), 3.529(dd, 1H, J=3.5, 13.7 Hz), 3.864 (s, 1H), 4.776 (dd, 1H, J=3.4, 11.6 Hz),6.432 (d, 1H, J=8.2 Hz), 6.89 (dd, 1H, J=2.1, 8.3 Hz), 7.330 (d, 1H,J=2.1 Hz), 7.552 (s, 1H), 7.914 (s, 1H).

Example 2

Inhibitory Action of the Compound of the Present Invention AgainstBiosynthesis of Brassinosteroid

Several Arabidopsis mutants having defects in the brassinosteroidbiosynthetic pathway have been isolated, and their common morphologicalcharacteristic has been revealed as dwarfism. Arabidopsis mutants suchas det2 (de-etiolation) and cpd (constitutive photomorphogenesis anddwarfism) show strong dwarfism manifested as curled leaves in deepgreen. Although this phenotype disappears when brassinolide isadministered externally, other plant hormones such as IAA andgibberellin are ineffective. Based on these findings, the compound ofthe present invention (Compound 1 obtained in Example 1) was used inArabidopsis (wild-type, Colombia) germination assay. Screening wasperformed under a light condition with criteria whether or not thecompound generates abnormality 1) which is morphologically similar tomutants in the brassinosteroid biosynthesis and 2) which disappears whenbrassinolide is administered. It was also examined whether or not themorphological change induced by the test compound was restored to themorphology of wild-type by addition of braassinolide under a darkcondition.

The Arabidopsis germination assay was performed as follows. Seeds ofwild-type and det were treated at a low temperature (2° C.) for 2 days,and the surfaces were sterilized with 1% NaOCl solution for 2 minutesand washed seven times with sterilized distilled water. The seeds weresown on 1% agar solid medium containing 0.5×Murashige and Skoog (1962)salt and 1.5% (w/v) sucrose in a plastic plate in the presence orabsence of the test compound. The wild-type plant and det2 plant weregrown in a growth chamber for 16 hours under a light condition (240mE/m2s) and for 8 hours under a dark condition (25° C.). For thescreening experiment, the plate was sealed with Parafilm (AmericanNational Can Co., Ltd., Chicago, Ill., USA). For the restorationexperiment that required a longer experimental period, the seeds weresown on 1% agar solid medium containing 0.5×Murashige and Skoog (1962)salt and 1.5% (w/v) sucrose in Agripot (Kirin Brewery Co., Ltd.). Theplants were grown in a growth chamber for 16 hours under a lightcondition (240 mE/m2s) and for 8 hours under a dark condition (28° C.).

As a result, induction of deformed seedlings that were morphologicallysimilar to the mutants in the brassinosteroid biosynthesis was observedwith the compound of the present invention. Among the test compounds,Compound 1 (also referred to as “RMB-61” in this example) showed themost potent induction of dwarfism at 10⁻⁶ M. However, this effect wasnegated by simultaneous administration of brassinolide. The results ofdose-effect test of RMB-61 revealed that the shapes of the plants cameto significantly resemble to those of det2 mutant at a concentration of10⁻⁵ M or higher. Therefore, in the following experiments, RMB-61 wasused and characterized as an inhibitor of the brassinosteroidbiosynthesis.

Generally, the biological activity of brassinosteroids is oftenevaluated by the in vitro rice leaf lamina inclination bioassay(explantation method: Wada et al., Plant Cell Physiol., 22, pp.323-325,1981; Takatsuto, J. Chromat. A., 658, pp.3-15, 1994) which is highlysensitive and reliable. However, this conventional method is notsuitable for analyzing the mechanism of action of brassinosteroidbiosynthesis inhibitors. Recently, Fujioka et al. reported a method(whole plant method) in which the rice leaf lamina inclination bioassayis improved as an in vitro system. This method reflects the actualbiosynthesis process of brassinosteroids by administration of variousprecursors of brassinolide. The inhibitory action of the compound of thepresent invention against the brassinolide biosynthesis was evaluated bythe explantation method and the whole plant method.

The rice leaf lamina inclination bioassay was performed essentiallyaccording to the procedure reported by Fujioka et al. as mentioned aboveby using a dwarfed rice plant (Oryza sativa cv. Tan-ginbozu). Seeds wereimmersed in water for 2 days at 28° C., and germinated seeds weresubjected to selection of those having a uniform coleoptile length(about 1 to 2 mm). Five germinated seeds were planted on 30 ml of 1%agar medium contained in a glass jar (inner diameter: 26 mm×60 mm), andincubated under the aforementioned conditions for 3 days. Each testcompound for the bioassay was dissolved in ethanol and placed on alamina in an amount of 0.5 ml by using a microsyringe. After the plantswere incubated under the same growth conditions for 2 days, the externalangle between the leaf lamina and its sheath was measured by using anannular protractor. Thirty plants in total (6 jars) were used for eachtreatment.

As a result, absolutely no inhibition of the action of brassinolide byRMB-61 was observed in the explantation method (FIG. 1). Further, in thewhole plant method, RMB-61 inhibited the action of cathasterone, whichwas a precursor of brassinolide, whilst failed to offset the inclinationaction by teasterone, typhasterol and castasterone (FIG. 2). Theseresults indicate that RMB-61 inhibited the biosynthetic process ofbrassinosteroid at conversion step from cathasterone to teasterone, andthus stopped the supply of brassinolide in the plants.

In order to confirm the action of RMB-61 also in the Arabidopsis assay,the seeds were grown with addition of the aforementioned brassinolideprecursors, and recovery of the growth retardation caused by the RMB-61treatment was observed. Recent researches on the brassinosteroidbiosynthesis in Arabidopsis have revealed that the route starting fromcampesterol and reaching to castasterone via cathasterone, teasteroneand typhasterol constitutes a main pathway accompanied by C-6 oxidationin the early stages of the biosynthesis. Thus, it has been revealed thatmutations in these stages can be considered as essential causes ofgeneral phenotypes with brassinolide deficiency (Szekeres et al., Cell,85, pp.171-182, 1996). If RMB-61 is a specific inhibitor against theoxidation of cathasterone, it is expected that the morphological traitsof the treated plants have similarities to the phenotypes of the mutantssuch as cpd (Szekeres et al., Cell, 85, pp.171-182, 1996). It is furtherexpected that the action of RMB-61 will disappear when typhasterol,castasterone or brassinolide is administered, and in addition, the cpdphenotypes caused by brassinosteroids will disappear.

Teasterone, 3-deoxoteasterone, typhasterol, castasterone andbrassinolide strongly inhibited the action of RMB-61 in Arabidopsis.However, the growth retardation caused by RMB-61 was maintained in thepresence of cathasterone. Similar results were also obtained in theadministration experiment under a dark condition. In addition to thegrowth retardation, RMB-61 promoted greening of plants under weak light.This effect was also eliminated by administration of the aforementionedbrassinosteroids except for cathasterone.

Further, rice growth bioassay was performed as follows. Seeds of rice(Oryza sativa cv. Koshihikari) were immersed in tap water for 2 hours,and then 10 seeds were immersed in 2 ml of 10 mM sodium acetate buffer(pH 5.4) containing 10 mM CaCl₂ filled in a sterilized 15-ml test tubein the presence or absence of a test compound. The plants were grown ina growth chamber for 16 hours under a light condition (240 mE/m2s) andfor 8 hours under a dark condition (25° C.). The plants were collectedand the length of shoots was measured.

Uniconazol, which is a gibberellin biosynthesis inhibitor, showed stronggrowth retardation action for the rice plant based on inhibitory actionagainst the gibberellin biosynthesis, whilst RMB-61 had no effect on thegrowth of the rice plant. On the other hand, when teasterone orbrassinolide was added to the rice plant in which the effect ofcathasterone was negated by RMB-61, the response of the rice plant tothe brassinosteroid was restored. This results well accorded with theaforementioned results. Further, RMB-61 completely failed to effect thegibberellin treatment, and the results suggest that the target site ofRMB-61 is essentially different from that of uniconazol orpaclobutrazol, which are strong inhibitors of the gibberellinbiosynthesis.

The materials used for the aforementioned methods are as follows.

Plant materials

The de-etiolated 2 (det2) mutant was obtained by uniquely mutatingwild-type seeds of the Colombia ecotype and isolating the mutant (Choryet al., Plant Cell, 3, pp.445-459, 1991; Li et al., Science, 272,pp.398-401, 1996). The wild-type seeds (Colombia) were purchased fromLHELE Seeds (Round lock, Tex., USA). The seeds of rice (Oryza sativa cv.Koshihikari and Tan-ginbozu) were presented by Dr. I. Honda (NationalAgriculture Research Center, Tsukuba, Japan).

Test compounds

Brassinolide was purchased from CID-tech Research Inc. (Ontario,Canada). Cathasterone and castasterone were synthesized as describedabove. Murashige and Skoog salt and vitamin mixture were purchased fromGIBCO BRL (Gland Island, N.Y., USA).

As explained above, the morphological change induced by the compound ofthe present invention disappears when exogenous brassinolide isadministered, and the brassinosteroid biosynthesis process, which wasinhibited by the compound of the present invention, was found to be astage preceding the teasterone synthesis. Furthermore, the compound ofthe present invention did not show growth inhibitory action in the riceelongation test, in which elongation retardation action by paclobutrazolwas observed which is a gibberellin biosynthesis inhibitor having asimilar chemical structure. Accordingly, it was revealed that thecompound of the present invention was a specific inhibitor against thebrassinosteroid biosynthesis.

Industrial Applicability

The compounds of the present invention have a specific inhibitory actionagainst the brassinosteroid biosynthesis, and are useful as activeingredients of plant growth regulators and so forth.

What is claimed is:
 1. A compound represented by the following formula:

wherein R¹ represents a lower alkyl group, R² represents a phenyl groupwhich is substituted or unsubstituted or a lower alkyl group, and R³represents a phenyl group which is substituted or unsubstituted, or asalt thereof.
 2. The compound or a salt thereof according to claim 1,wherein R¹ is methyl group or ethyl group, and R² represents a phenylgroup which is substituted or unsubstituted, or tert-butyl group. 3.4-(4-Chlorophenyl)-2-phenyl-3-(1,2,4-triazoyl)-butan-2-ol or a saltthereof.
 4. An inhibitor against the brassinosteroid biosynthesis whichcomprises the compound according to claim 1 a salt thereof as an activeingredient.
 5. An inhibitor against the brassinosteroid biosynthesiswhich comprises the compound according to claim 2 or a salt thereof asan active ingredient.
 6. An inhibitor against the brassinosteroidbiosynthesis which comprises the compound according to claim 3 or a saltthereof as an active ingredient.